Single particle cryo-EM has achieved great success in recent years as a versatile structural analysis tool that does not require crystals. It is routinely used to study the structures of large biological assemblies, including HIV protein complexes that are intractable by crystallography and NMR. However, the use of EM to study small proteins and integral membrane proteins is limited by the current technology, despite tremendous needs for these applications. We aim to develop novel approaches to overcome these limitations and to apply high-resolution single particle cryo-EM to study membrane and soluble HIV proteins. For cryo-EM, proteins are captured in their native conformations by embedding them in vitreous ice at liquid nitrogen temperature, followed by imaging at low, non-damaging electron doses. Thousands of particle images are aligned and averaged to calculate 3D reconstructions. This approach has achieved near atomic resolution for large protein assemblies with high symmetry, such as non-enveloped icosahedral viruses [131- 133], and now even for complexes without symmetry, such as mammalian chaperonins [134] and the ribosome [135]. Obtaining high-resolution EM structures depends critically on sample homogeneity and the ability to accurately align individual images. In general, large molecules are easily recognized in noisy images and often have sufficiently well-defined features to facilitate image alignment [136]. So far, all high-resolution structures determined by this method are from large molecular complexes, typically with molecular weights in the mega- Dalton range. Single particle cryo-EM has also been applied to study smaller proteins and complexes, although it usually generates only low-resolution structures. It is generally acknowledged to be very difficult to obtain 3D reconstructions better than 20 A for proteins smaller than 200 kDa, and nearly impossible for proteins smaller than 100 kDa [136]. However the need for this application is enormous, particularly for HIV complexes that often have flexible or disordered portions and consequently are difficult to crystallize in their native states. In practice, negative stain single particle EM can be used to determine structures of relatively small proteins, but to resolutions lower than 20 A [137]. We have made great recent strides towards overcoming these limitations and will continue methods development and their application to HIV-host complexes.